AAPS PharmSciTech, Vol. 13, No.

4, December 2012 ( Ⓒ 2012)

DOI: 10.1208/s12249-012-9847-7

Research Article

Product Development Studies on Surface-Adsorbed Nanoemulsion

of Olmesartan Medoxomil as a Capsular Dosage Form

Sumita Singh,1 Kamla Pathak,1 and Vikas Bali1,2

Received 8 May 2012; accepted 22 August 2012; published online 11 September 2012
Abstract. The present study aimed at development of capsular dosage form of surface-adsorbed nano-
emulsion (NE) of olmesartan medoxomil (OLM) so as to overcome the limitations associated with
handling of liquid NEs without affecting their pharmaceutical efficacy. Selection of oil, surfactant, and
cosurfactant for construction of pseudoternary phase diagrams was made on the basis of solubility of drug
in these excipients. Rationally selected NE formulations were evaluated for percentage transmittance,
viscosity, refractive index, globule size, zeta potential, and polydispersity index (PDI). Formulation (F3)
comprising of Capmul MCM® (10% v/v), Tween 80® (11.25% v/v), polyethylene glycol 400 (3.75% v/v),
and double-distilled water (75% v/v) displayed highest percentage cumulative drug release (%CDR;
96.69± 1.841), least globule size (17.51±5.87 nm), low PDI (0.203±0.032), high zeta potential (−58.93±0.98
mV), and hence was selected as the optimized formulation. F3 was adsorbed over colloidal silicon dioxide (2
ml/400 mg) to produce free-flowing solid surface-adsorbed NE that presented a ready-to-fill capsule
composition. Conversion of NE to surface-adsorbed NE and its reconstitution to NE did not affect the in
vitro release profile of OLM as the similarity factor with respect to NE was found to be 66% and 73%
respectively. The
%CDR after 12 h for optimized NE, surface-adsorbed NE, and reconstituted NE was found to be 96.69±0.54,
96.07 ±1.76, and 94.78±1.57, respectively (p>0.05). The present study established capsulated surface-
adsorbed NE as a viable delivery system with the potential to overcome the handling limitations of NE.
KEY WORDS: bioavailability; nanoemulsion; olmesartan medoxomil; oral.

INTRODUCTION pump and thus augment the intestinal absorption of therapeu-

Olmesartan medoxomil (OLM) is an extensively used
antihypertensive drug (1). By the action of aryl esterases,
situated in both intestine and plasma, it gets quickly de-esteri-
fied upon oral administration in to an active metabolite, i.e.,
olmesartan (2). Olmesartan is reported to be more helpful in
patients with essential hypertension in comparison to other
angiotensin II receptor blockers with respect to decrease in
ambulatory blood pressure (3). Commercially available tab-
lets of OLM exhibit reduced oral absorption leading to low
oral bioavailability of 25.6% (4). OLM is highly lipophilic
in nature (log p=4.31) which attributes to its low aqueous
solu- bility. Poor aqueous solubility and efflux of hydrophobic
ther- apeutic agents by means of drug resistance pump in
the gastrointestinal tract contribute to their low
bioavailability (5,6). Oil to water nanoemulsion systems are
reported to augment aqueous solubility of hydrophobic
therapeutic agents by including them in the oil phase of the
nanoemulsion (7–9). Non-ionic surfactants such as Tween 80
have been stated to be valuable pharmaceutical excipient in
order to prevent the function of the drug-resistant P-
glycoprotein (P-gp) efflux
1
Department of Pharmaceutics, Rajiv Academy for Pharmacy,
Mathura, 281001Uttar Pradesh, India.
2
To whom correspondence should be addressed. (e- mail:
vksbali@gmail.com)
tic agents susceptible to P-gp-mediated efflux in the intestine
(10).
OLM has been formulated as a self-microemulsifying
drug delivery system (SMEDDS) using capryol 90, tween 20
and tetraglycol (10:60:30, v/v/v) and Lee and his coworkers
reported an improved relative bioavailability of 170% com-
pared to the suspension in male Sprague–Dawley rats (11).
SMEDDS as a delivery system have been reported to employ
high concentration (30–60%) of surfactants that may lead to
cellular toxicity (12). Furthermore, precipitation of the hydro-
phobic therapeutic agent formulated as SMEDDS and encap-
sulated in gelatin capsule may occur due to propensity of the
volatile solvents used in formulation of SMEDDS to get trans-
ferred to the shell (13). One of the approaches to circumvent
these limitations is to deliver hydrophobic therapeutic agents
in the form of a nanoemulsion formulation that like SMEDDS
is also isotropic, thermodynamically stable, transparent (or
translucent) system of oil, water, and surfactants. Nanoemul-
sion (NE) possesses droplet size generally in the range of 10–
100 nm but in contrast to SMEDDS, it employs a lesser
amount (5–10%) of surfactant. Furthermore, NEs can be
formulated with little energy input (heat or mixing), have a
long shelf life, and is characterized with simplicity of scale up
of the manufacturing process (14). Hence, it was hypothesized
that developing a nanoemulsion-based drug delivery system
utilizing a non-ionic surfactant as the emulsifier would help to

1530-9932/12/0400-1212/0 Ⓒ 2012 American Association of Pharmaceutical Scientists 1212

Surface-Adsorbed NE of Olmesartan Medoxomil
improve solubility and prevent efflux of OLM out of the
intestine eventually leading to enhanced oral bioavailability.
Although NE is one of the finest modes of delivery for
hydrophobic therapeutic agents, but due to liquid nature of the
dosage form, it would not be as accepted as the solid dosage
form. Liquid dosage forms are normally associated with trans-
portation issues, instability problems, and poor palatability due
to the lipid content (in case of NE). Moreover, as potent drugs
are incorporated in NE formulation, dose variability due to
handling problems in case of select patient population may
lead to toxicity. To manage these challenges, many attempts
have been made to convert liquid formulations into the solid
dosage forms like capsule (15–17) and tablet (18).
Based on these considerations, the current study was
aimed at developing and characterizing NE of OLM so as to
improve dissolution rate-limited absorption of the drug
using a low surfactant concentration and to convert the
developed liquid NE into a solid unit dosage form by
adsorbing it over an inert, solid adsorbent so as to develop a
patient friendly dos- age form.
MATERIALS
OLM was obtained as a gift sample from Sun Pharma-
ceutical Industries (Sikkim, India). Capmul MCM® (glycerol
monodicaprylate) and Captex 100® were obtained as gift
samples from Abitech Corporation Limited (Janesvile, WI,
USA). Labrafil M 1944 CS® (oleoyl macrogoglyceride), Lab-
rafil M 2125CS® (linoleoyl macrogolglycerides), Labrasol®
(caprylo caproyl macrogol-8-glyceride), Peceol® (glyceryl
ole- ate), Plurol oleique® (polyglycerol oleate), Lauroglucol
90® (propylene glycol monolaurate), and Transcutol® P
(diethy- lene glycol monoethyl ether) were donated by
Gattefosse (Saint Priest, Cedex, France). Tween 80®
(polyoxyethylene sorbitan monooleate), Tween 20®
(polyoxyethylene sorbitan monolaurate), ethanol, and
polyethylene glycol 400 (PEG 400) were obtained from S.D.
Fine-Chemicals Ltd. (Mumbai, India). Colloidal silicon
dioxide and dialysis membrane (pore size of 25 Å) were
obtained from Hi Media Laboratories Pvt. Ltd (Mumbai,
India). All other chemicals and solvents were of analytical
reagent grade and were used without further purification.
METHODS
Screening of Components
Solubility of OLM in various vehicles (oils, surfactants,
and cosurfactants) was determined by shake flask method
whereby an excess amount of drug was added to 2 ml of the
selected vehicle and kept at 25±1°C in an isothermal bath
shaker (Hicon, New Delhi, India) for 72 h to reach
equilibri- um. The samples were centrifuged (Remi Pvt.
Ltd., Vasai, India) at 3,000 rpm for 15 min. Supernatant
was removed carefully and diluted suitably with ethanol
(95%, v/v). The samples were analyzed for drug content at
257.8 nm using UV- visible spectrophotometer (Shimadzu,
Pharmaspec1700, Kyoto, Japan). The solubility of drug in
each component was calculated in triplicate and
mean±standard deviation (SD) was reported.
1213
Construction of Pseudoternary Phase Diagram
On the basis of solubility study of drug, Capmul MCM
was chosen as the oil phase, Tween 80 as the surfactant, and
PEG 400 as the cosurfactant. Double-distilled water was used
as the aqueous phase for the creation of pseudoternary
phase diagram. Surfactant and cosurfactant were mixed
(Smix) in different volume ratios (1:1, 1:2, 1:3, 2:1, 3:1, 4:1).
For each phase diagram, oil and specific Smix ratio was
mixed in differ- ent volume ratios from 1:9 to 9:1 so that
maximum ratios were enclosed for the study in order to
define the boundaries of phases formed in the phase
diagram. Pseudoternary phase diagrams were developed by
aqueous titration data using PCP Disso V2.08 software,
Pune, India. Physical state of the blank NE was marked on a
pseudo-three-component phase diagram in which one axis
signified the aqueous phase, the second signified oil, and
the third signified Smix. A total of 14 formulations were
selected from the NE regions of the phase diagram.
Evaluation of Blank NEs
Thermodynamic Stress Stability Studies
Blank NE formulations were centrifuged at 3,500 rpm
for 30 min and monitored for phase separation, creaming, or
crack- ing. Those formulations that did not show any phase
separation were subjected to heating–cooling cycle. Six cycles
between re- frigerator temperature (4°C) and 45°C with
storage at each tem- perature for not less than 48 h were
performed. The formulations that passed heating–cooling
cycles were subjected to three freeze–thaw cycles at
temperature between −21°C and +25°C with storage at each
temperature for not less than 48 h. The formulations that
passed the thermodynamic stress stability tests were further
taken for dispersibility study in order to estimate the efficiency
of emulsification.
Dispersibility Test
The efficiency of emulsification of oral NE was assessed
using a standard USP XXII dissolution apparatus II. One
milliliter of each formulation was added to 500 ml of double-
distilled water at 37±0.5°C. A standard stainless steel dissolu-
tion paddle rotating at 50 rpm presented gentle agitation. The
in vitro behavior of the formulation was visually assessed
using the grading system. Grade A was given to the
formulations which exhibited clear or bluish appearance within
1 min, grade B included formulations with slightly less clear
and bluish white appearance, grade C was given to the
formulations with fine milky appearance within 2 min, grade D
was assigned to the formulations with dull, grayish white
having slightly oily appearance in more than 2 min, and graded
E were the for- mulations with large oil globules present on the
surface. For- mulations, from each Smix ratio investigated, were
selected on the basis of least Smix and suitable amount of oil
(0.5 ml) that could completely dissolve the required amount (5
mg) of drug. Care was taken to select the formulations which
passed the thermodynamic stress stability and dispersibility
test in grades A and B.
1214
Preparation of Drug-Loaded NE
Thermodynamically stable blank NEs were chosen for
drug loading. Drug-loaded NEs were prepared by adding the
calculated amount of drug (10.02 mg/ml of OLM) to the oil
phase and stirring in isothermal bath shaker until whole of the
drug was dissolved. Then, Smix in a fixed proportion was
added to fixed volume of oil containing drug to produce a
clear mixture. This was followed by adding definite proportion
of water (drop wise) and shaking slowly until a clear NE was
obtained.
Characterization of the NE
Globule Size, Zeta Potential, and Polydispersibility Index
Globule size of the NEs was determined by photon cor-
relation spectroscopy that analyzes the fluctuation in light
scattering due to Brownian motion of the globules, using a
Zetasizer ver. 6.01 (Malvern Instrument Ltd., UK). The for-
mulation was subjected to 500 times dilution with double-
distilled water and light scattering was monitored at 25°C at
a 90° angle. Zeta potential was also measured using the
same instrument. The refractive index was kept at 1.33 and
viscosity at 1.0 cps to mimic the values for pure water. Zeta
potential values were determined from the electrophoretic
mobility of oil droplets.
Percentage Transmittance, Refractive Index, and Viscosity
Percentage transmittance was determined spectrophoto-
metrically. One milliliter of the formulation was diluted to 100
times with double-distilled water and percentage transmit-
tance was measured against double-distilled water as blank
at 630 nm. Abbe’s type refractrometer (Jindal Instruments,
Ambala, India) was used to determine the refractive index.
Few drops of NE were smeared on the lower prism surface.
The eye piece cross-wire was adjusted so that a sharp demar-
cation line passes through the center having half light and half
dark position. Then, readings were noted down from the scale.
The viscosity of the formulations was determined without
dilution using Brookfield viscometer DV-II + Pro (Brookfield
Engineering Laboratories, Inc, MA, USA) coupled with S-94
spindle at 100 rpm and 25±2°C.
Drug Content
For the determination of drug content, 1 ml of the NE
was taken in a 10 ml volumetric flask and shaken
vigorously with ethanol (95%, v/v) for 10 min. Finally, the
volume was made up to 10 ml with the same solvent and
after proper dilution using ethanol, the drug content was
analyzed at
257.8 nm by UV spectrophotometer (Shimadzu, Pharmas-
pec1700, Kyoto, Japan).
In Vitro Drug Release
In vitro drug release was measured by dialysis bag
meth- od using a pretreated dialysis membrane (MWCO 12–
14 kD). Himedia dialysis membrane (Himedia Laboratories
Pvt. Ltd., Mumbai, India) was kept in a normal saline
solution for 2 h
Singh, Pathak and Bali
before study to ensure complete wetting of the membrane.
Two milliliters of optimized formulations was placed in pre-
treated dialysis bag and drug release was studied using USP
dissolution apparatus II (Hicon Enterprises, Delhi, India)
containing 500 ml of phosphate buffer (pH7.4) at 37±0.5°C.
The speed of the paddle was adjusted to 50 rpm. Two-milliliter
sample was withdrawn at regular time intervals (0, 0.5, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, and 12 h) and the volume withdrawn
was replaced with the fresh medium. The release of drug from
the NE formulations was compared against the pure drug
suspen- sion. The samples were analyzed at 257.8 nm and the
percent- age cumulative drug release (%CDR) was calculated.
The analysis of the samples was done in triplicate.
Transmission Electron Microscopy
Morphology of the oil droplets in the NE formulation was
visualized using CM 10 transmission electron microscope
(Mega View III FW, Philips, UK). NE formulation was diluted
100 times and a drop was applied to 300 mesh copper grid.
The grid was inverted and a drop of phosphotungstic acid
(PTA) was applied to the grid for 10 s. Excess of PTA was
removed and grid was analyzed at 60–80 kV.
Selection of Optimized NE
Optimized formulation was selected on the basis of glob-
ule size, zeta potential, polydispersity index (PDI), percent
drug content, and %CDR. The optimized NE was utilized
for the preparation of surface-adsorbed NE by adsorption
over a solid adsorbent.
Adsorption of NE Over a Solid Adsorbent
The optimized NE, F3 (5 ml) was placed in a glass mortar
and colloidal silicon dioxide (400 mg) was added slowly and
mixed gently to get the solid mass. The solid mass was passed
through the sieve (22 mesh size) to get uniform free-flowing
powder. The powder was stored over anhydrous calcium chlo-
ride in a dessicator until further evaluation.
Micromeritic and Rheological Characterization of Surface-
Adsorbed NE
The flow properties of the surface-adsorbed NE were
determined by the following tests: (1) Carr’s compressibility
index, (2) Hausner’s ratio, and (3) angle of repose. The sur-
face-adsorbed NE (5 g) was poured lightly into 50 ml measur-
ing cylinder. The powder was subjected to tapping until no
further change in volume was observed. Bulk density (Do) and
tapped density (Df) of the powder was calculated by dividing
the weight of the granules by its volume before and after
tapping, respectively. Percentage compressibility was comput-
ed by subtracting Do from Df and divided by Df and multiplied
by 100, Hausner’s ratio was computed as Df divided by Do.
Angle of repose was measured by static funnel method. Angle
of the heap of granules (5 g) formed by passing through a
funnel placed at a height of 8 cm from the horizontal surface
was measured using a protractor (17).
Surface-Adsorbed NE of Olmesartan Medoxomil
Drug Content Determination
Surface-adsorbed NE equivalent to 5 mg of OLM was
dispersed in suitable quantity of ethanol (95%, v/v). The
sample was mixed thoroughly to ensure complete dissolution
of drug in ethanol. The sample was centrifuged using centri-
fuge (Remi Pvt Ltd., Vasai, India) at 3,500 rpm for 15 min to
separate colloidal silicon dioxide particles. The supernatant
was suitably diluted and analyzed spectrophotometrically at
257.8 nm. Drug content was computed from the validated
calibration curve of drug in ethanol (95%, v/v).
Scanning Electron Microscopy
The morphological features of particles of colloidal sili-
con dioxide and surface-adsorbed NE were investigated by
JEOL-5400 (Tokyo, Japan) scanning electron microscope.
Gold sputter coating of all the samples was done to render
the surface of particles electroconductive. The micrographs
were viewed at ×100 and ×500 magnifications.
Assessment of Reconstituted NE
The surface-adsorbed NE powder (420 mg) was resus-
pended in 3.75 ml double-distilled water and shaken gently for
5 min. The reconstituted NE (RF3) was characterized for
globule size and zeta potential using Zetasizer ver. 6.01 (Mal-
vern Instrument Ltd., UK) as described earlier. The globule
size was visualized by TEM as detailed previously.
In Vitro Drug Release
In vitro drug release study of surface-adsorbed NE was
performed by introducing the powder equivalent to 5 mg drug
into the release test media using USP dissolution apparatus
II (Hicon Enterprises, Delhi, India) with paddle rotation
of 50 rpm. The dissolution media consisted of 500 ml of
phos- phate buffer, pH7.4 maintained at 37±0.5°C and
samples were withdrawn at predetermined time intervals.
The amount of drug release was estimated by measuring
absorbance of the samples at 257.8 nm and the release
profiles of surface- adsorbed NE and RF3 were evaluated
for similarity against the release profile of F3 (optimized
NE).
Statistical Analysis
The results were expressed as mean±SD and were ana-
lyzed statistically by one-way analysis of variance (ANOVA)
using Graph Pad Prism V5.04 software (San Diego, CA,
USA).
RESULTS AND DISCUSSION
Screening of Components
Solubility of the therapeutic agent in the oil phase of
nanoemulsion governs the ability of the nanoemulsion formu-
lation to preserve the drug in solubilised form during its shelf
life and after oral administration. Hence, the solubility of the
therapeutic agent in oils, surfactants, and cosurfactants was
the most significant criterion for the screening of NE compo-
nents. OLM is hydrophobic and less polar in nature that was
1215
confirmed by its poor solubility of 0.0071 mg/ml in water
(polar solvent). However, the solubility of OLM was higher
in nonpolar solvents. Consequently, the solubility of OLM was
found to be maximum (10.023 ± 1.517 mg/ml) in Capmul
MCM, a medium chain mono/diglyceride (Fig. 1). Higher
solubility of drug in Capmul MCM may be attributed to the
nonpolar nature of the poorly water-soluble drugs that sup-
ports their solubilization in oils like medium chain triglycer-
ides or mono- or diglycerides (7). High solubility of drug in oil
is particularly advantageous in NE formulation. The higher
the solubility of the drug in oil phase, the lower will be the
volume of oil required to dissolve the single dose of drug. As a
result, less quantity of surfactant and cosurfactant may be
required for NE formulation. In addition to oil, the solubility
of drug in the surfactant is also important. The solubility of
OLM was highest in Tween 80, among the examined surfac-
tants and PEG 400 among the examined cosurfactants. Thus,
for the formulation of NE, Capmul MCM, Tween 80, and
PEG 400 were selected as oil phase, surfactant, and cosurfac-
tant, respectively.
Construction of Pseudoternary Phase Diagrams
The study of phase behavior helps to precisely character-
ize a phase boundary. Knowledge about the boundaries of the
different phases as a function of composition variables can be
attained by preparing phase diagrams. Selection of oil, surfac-
tant, and the mixing ratio of oil to surfactant/cosurfactant
mixture are vital for the NE formation (7). Low toxicity,
resistance to pH, and ionic strength changes are some of the
attributes that favor utilization of nonionic surfactants for the
NE formulations. It was observed that when Tween80 (surfac-
tant) was used alone, a significant zone of NE was obtained
(Fig. 2a) but when PEG 400 (cosurfactant) was used along
with the surfactant in a ratio of 1:1 (Fig. 2b), a tremendous
decrease in the NE region was observed and 72.72% (v/v) of
oil could be emulsified using 16.66% (v/v) of Smix. However,
when the cosurfactant was utilized in 1:2 ratio (Fig. 2c), the
amount of oil that could be emulsified was 52.17% (v/v) using
34.78% (v/v) of Smix. On further increasing the proportion of
cosurfactant in the Smix to 1:3 (Fig. 2d), a decrease in NE
region was observed with the maximum amount of oil that
could be emulsified being reduced to 38.78% (v/v) using
52.17% (v/v) of Smix. With the Smix ratio of 1:4 (Fig. 2e),
further decrease in the NE region was observed and the
maximum amount of oil that could be emulsified was
36.36% (v/v).
In addition to varying cosurfactant in the Smix, the effect
of varying the concentration of surfactant in the Smix from
1:1 to 2:1 (Fig. 2f) resulted in considerable increase in the
NE region. On changing the concentration of surfactant in
Smix from 2:1 to 3:1 (Fig. 2g) and then to 4:1 (Fig. 2h), it
was observed that NE region did not change significantly. It
was also concluded that 63.63% (v/v) of oil could be
emulsified using 11.25% (v/v) of surfactant in comparison
to emulsifica- tion of 10% (v/v) of oil by using 60% (v/v) of
surfactant.
Selection of NEs from Phase Diagram
From the pseudoternary phase diagrams, 14 formulations
were selected to fulfill the following criteria
1216 Singh, Pathak and Bali

Fig. 1. Saturation solubility bar chart for selection of excipients for nanoemulsion of OLM
at 25±1°C

1. The proportion of oil used should be able to solubilize

the drug (single dose) completely. One milligram of
OLM is dissolved easily in 0.1 ml of oil.
2. The minimum concentration of the Smix used for that
amount of oil was taken.
3. The frequently used dose of the OLM is 5 mg (19).
Therefore, 5 mg was selected as the dose for the de-
velopment of NE formulation.
4. For convenience, 2 ml was selected as the volume of
the NE formulation, so that it can be increased or
decreased as per the requirement.
Thermodynamic Stress Stability
Thermodynamically stability of nanoemulsion formula-
tions differentiates them from emulsions that have kinetic
stability which, NEs by virtue of their thermodynamic stability
do not exhibit phase separation, creaming, or cracking unlike
emulsions that finally phase separate (20). Thus, the selected
formulations were subjected to different thermodynamic sta-
bility tests such as centrifugation, heating–cooling cycles, and
freeze–thaw cycles (Table I). The formulations that passed
these tests were selected for the dispersibility study to evalu-
ate the efficiency of emulsification.
Dispersibilty
Gastrointestinal (GI) fluid is responsible for the
dilution of oral NE and results in the gradual desorption of
surfactant located at the oil–water interface. This process is
thermody- namically governed by the tendency of the
surfactant to pre- serve its concentration to its critical
micelle concentration. In order to evaluate the
emulsification efficiency of NE formula- tions upon infinite
dilution in GI fluid, double-distilled water was used as a
dispersion medium. This selection was based on the report
corroborating insignificant difference in the behavior of NE,
prepared using nonionic surfactants, dispersed in either water
or simulated gastric or intestinal fluid (21,22). The formu-
lations that passed the dispersibility test in double-distilled
water

Fig. 2. Pseudoternary phase diagrams involving Capmul MCM, Tween 80, and PEG 400 as the oil, surfactant, and cosurfactant, respectively.
Ratio of surfactant to cosurfactant in a is 1:0, b 1:1, c 1:2, d 1:3, e 1:4, f 2:1, g 3:1, and h 4:1. Marked area oil in water NE region
 Table I. Selection of Final Test Nanoemulsion Formulations by Thermodynamic Stress Stability Studies and Dispersibilty Test Su
rfa
Percentage v/v of NE components Observation ce
Recoded -
Formulation code Smix ratio Oil Smix Water Centrifugation Heating-cooling cycle Freeze-thaw cycle Dispersibilty Results formulations Ad
so
N1 1:0 10 40 50 ✓ ✓ ✓ Grade A Passed F1 rb
N2 1:0 10 38 52 ✓ ✓ Х Grade C Failed –
ed
N3 1:0 10 36 54 ✓ ✓ Х Grade C Failed –
N
N4 2:1 10 15 75 ✓ ✓ ✓ Grade A Passed F2
N5 2:1 10 42 48 Х – – Grade D Failed –
E
N6 2:1 10 40 50 Х – – Grade D Failed – of
N7 2:1 10 38 52 Х – – Grade D Failed – Ol
N8 2:1 10 37 53 Х – – Grade D Failed – m
N9 3:1 10 15 75 ✓ ✓ ✓ Grade A Passed F3 es
N10 3:1 10 43 47 ✓ Х – Grade C Failed – ar
N11 4:1 10 15 75 ✓ ✓ ✓ Grade B Passed F4 ta
N12 4:1 10 42 48 Х – – Grade D Failed – n
N13 4:1 10 40 50 Х – – Grade D Failed – M
N14 4:1 10 35 55 ✓ ✓ ✓ Grade B Passed F5 ed

✓ = Pass, Х = fail

Table II. Mean (±SD, n=3) Globule Size, Polydispersity Index (PDI), Zeta Potential, Drug Content, Refractive Index, Percentage Transmittance, and Viscosity of Final Test Formulations

Mean globule Mean zeta potential± Mean Drug content± Mean refractive Mean percentage Mean viscosity±
Formulation code size±SD (nm) Mean PDI±SD SD (mV) SD (%) index±SD transmittance±SD SD (cps)
F1 15.08±7.92 0.150±0.012 −33.51±0.21 85.66±1.42 1.332±0.004 88.67±0.13 291±0.78
F2 79.09±6.54 0.543±0.021 −48.72±0.92 89.40±2.04 1.389±0.002 92.75±0.64 286±0.34
F3 17.51±5.87 0.203±0.032 −58.93±0.98 95.60±1.23 1.334±0.003 99.78±0.23 254±0.35
F4 29.21±10.25 0.490±0.041 −58.32±1.2 91.77±3.21 1.374±0.001 98.74±0.08 293±0.21
F5 206.00±8.76 0.480±0.032 −56.51±1.09 86.24±2.72 1.362±0.013 96.88±0.05 319±1.04

12
17
1218
Fig. 3. In vitro drug release study of NE formulations in phosphate
buffer pH7.4 at 37±1±°C
in grade(s) A and B were selected for further study as
these formulations were certain to remain as NE upon
dispersion in the aqueous environment of the GI tract
(Table I). Selected formulations were taken for globule
size, zeta potential, PDI, refractive index, viscosity, per-
centage transmittance, drug content, and in vitro drug
release determinations.
Characterization of NE
Globule Size, Zeta Potential, and Polydispersibility Index
Drug release and subsequent absorption are largely in-
fluenced by the globule size of NE (23). Rapid diffusion of
drug from smaller droplets into aqueous phase favors drug
dissolution. Globule size of the prepared NE was
determined and results are shown in Table II. As observed,
formulation F1 had the smallest globule size (15.08±7.92 nm)
followed by F3 (17.51±5.87 nm). F1 formulation contained
higher Smix ratio (40%) as compared to F3 (15%) that
probably favored reduc- tion in globule size. Diameter of
the dispersed oil droplets of the NE was found to be much
smaller than the diameter of smallest blood capillary (400
nm). This is advantageous as it avoids any probability of
capillary blockage during transfer of the droplets in vivo. The
small size of the globules also favors long circulation time
(12,24)
Charge on the oil droplets in NE is another attribute that
should be investigated while studying the absorption of nano-
emulsion (25). Zeta potential also indicates NE stability with
Singh, Pathak and Bali
regards to degree of repulsion between adjacent, similarly
charged globules in NE. Conventionally, zeta potential can
be positive or negative in the range of −30 to +30 mV. The
globules of OLM NEs displayed negative value of zeta poten-
tial (Table II) that could be due to the presence of negatively
charged free fatty acids component of oil used in the formu-
lation of nanoemulsion. Maximum value of zeta potential of
−58.93±0.98 mV was recorded for F3 closely followed by F4
and F5, and least zeta potential was documented for F1.
Similar pattern was also observed for drug content determi-
nation and hence, it can be inferred that the amount of drug
entrapped in the NE globule also influenced the magnitude of
charge on the globules. The polydispersity index of F1 formu-
lation was least (0.150±0.012) followed by F3 formulation as
0.203±0.032. Lower value of PDI is favorable, as it ensures
uniformity in size of nanoemulsion globules.
Percentage Transmittance, Refractive Index, and Viscosity
The value of percentage transmittance of formulations F1
to F4 was closer to 100% indicating that the formulations were
clear and transparent (Table II). Among all the formulations,
F3 showed highest value of percentage transmittance (99.78±
0.23%) which was significantly (p<0.001) higher in compari-
son to other formulations. The refractive index that also meas-
ures transparency was 1.334±0.003 for F3 very close to that of
water (1.334) and ensured its homogeneous character. The
viscosity analysis revealed low viscosity for all formulations
and F3 showed minimum viscosity of 254±0.35 cps. Thus, F3
can be easily administered as a uniform dose.
In Vitro Drug Release
In vitro drug release profiles of OLM from NE formula-
tions (F1–F5) and pure drug suspension (S) are shown in
Fig. 3. The release of OLM from all the NE formulations
was higher than the release profile of S (47.38%±0.352 in
12 h). Maximum %CDR of 96.69 ± 1.841 was displayed by
formulation F3 followed by F4 (90.53%±3.311) in 12 h.
The reason for the differences in percentage cumulative
drug release of F3 and F4 can be correlated to the Smix
composition and its quantity in NE. The Smix in F3 was in
the ratio of 3:1whereas in F4 it was 4:1, both present in
15% by volume in respective NEs. As described in liter-
ature, an increase in surfactant concentration results in
decrease in the droplet size, but this phenomenon levels
off at a particular surfactant concentration whereby any
Table III. Interpretation of Drug
Release Pattern of Nanoemulsion
Formulations
r2 Value
Formulation code Zero order First order Higuchi Pe
F1 0.9723 0.9217 0.9143 0.8
F2 0.9876 0.8791 0.9163 0.8
F3 0.9854 0.8764 0.9747 0.9
F4 0.9882 0.8794 0.9643 0.9
F5 0.9458 0.9173 0.8976 0.9
S 0.9256 0.8769 0.9043 0.8
a
Enhancement in %CDR after 12 h with respect to suspension
Surface-Adsorbed NE of Olmesartan Medoxomil
Fig. 4. Scanning electron micrographs of a colloidal silicon dioxide and b surface-adsorbed NE
further increase in surfactant concentration results in a
raise in droplet size (26). Stabilization of oil droplets by
virtue of decrease in droplet size can be attributed to the
localization of surfactant monolayer at the oil–water inter-
face (27). However, further rise in surfactant concentra-
tion leads to increased penetration of water into oil
droplets causing breakdown of oil droplets and resulting
in bigger droplets (28). Consequently, F4 had a larger
globule size of 29.21 ± 10.25 nm in comparison to 17.51
±
5.87 nm of F3 formulation that offered a larger surface
area for the release of OLM. In F5, very high percentage
of Smix (35%, v/v) resulted in large globules (206 ±
8.76 nm) that offered low surface area and consequently
low %CDR of 72.16% in 12 h. In F1 and F2, low Smix
ratios resulted in larger-sized globules and hence poor
release than F3. Modeling of the in vitro release data
indicated zero order as the best-fit model (Table III).
On applying one-way ANOVA followed by Dunnett’s test,
a significant difference was observed in the release pro-
files of F2, F4 (p <0.05), and F3 (p< 0.01) in comparison
to S (drug suspension).
Selection and Development of Optimized Formulation
Formulation F3 with highest %CDR of 96.69±1.841, least
globule size (17.51±5.87 nm), lower PDI value (0.203±0.032),
and high zeta potential (−58.93±0.98 mV) was selected as the
optimized formulation and was considered as stable and
homogeneous formulation. Transmission electron micros-
copy of F3 revealed dark and spherical spots against a
light background and the droplet size revealed by TEM
was in conformity with the zeta sizing results. Although
NE is one of the finest mode of delivery for hydrophobic
therapeutic agent OLM, but due to liquid nature of the
dosage form, it is normally associated with transportation
Table IV. Comparative Characterization of Reconstituted Nanoemulsion (RF3) and Nanoemulsion
(F3)
Formulation code Mean globule size±SD (nm) Mean PDI±SD
RF3 28.98±0.98 0.332±1.35
F3 17.51±5.87 0.203±0.03
1219
issues, instability problems, and poor palatability due to
the lipid content (in case of NE). Moreover, dose vari-
ability due to handling problems in case of select patient
population may lead to toxicity. To manage these chal-
lenges, liquid NE can be developed as a solid dosage form
by adsorbing on a highly porous solid with sufficient
adsorbing capacity and convert it to a free-flowing pow-
der. Colloidal silicon dioxide, a highly porous solid with a
specific surface area of 200–400 m2/g was selected (29) for
the development of capsular dosage form of surface-
adsorbed NE.
Characterization of Surface-Adsorbed NE
Micromeritic and Rheological Characterization
The bulk density of surface-adsorbed NE was found
to be 0.610 ± 0.103 g/cm3 and the tapped density was
0.7812 ± 0.052 g/cm3. Close values of bulk and tapped
density will ensure uniform filling of capsule shell at
industrial scale. The Hausner’s ratio derived from bulk
and tapped density was found to be 1.287 ± 0.572. A
Hausner’s ratio of less than 1.25 indicates good flow
property of the granules (30) and slightly higher value of
Hausner’s ratio can be improvised by use of flow
activators. The angle of repose that was 31.79°± 0.54,
categorized the flow as passable (31) reconfirming the
results of Hausner’s ratio. Carr’s compressibility index
was found to be 28.073 ± 1.782% indicating compressible
nature of the surface-adsorbed NE that presents an
opportunity for tabletting of the surface-adsorbed NE.
All these results proved that the surface-adsorbed NE
powder can be aptly filled in the hard gelatin capsule as
a solid unit dosage form.
Mean zeta potential±SD (mV) Mean drug content±SD (%)
−35.90±0.45 92.77±2.16
−58.93±0.98 95.60±1.23
1220 Singh, Pathak and Bali

Fig. 5. Transmission electron micrographs of a optimized nanoemulsion (F3) and b recon-

stituted nanoemulsion (RF3)

Scanning Electron Microscopy Dosage Form Development of Surface-Adsorbed NE and its

Evaluation
Colloidal silicon dioxide appeared to be spherical
particles approximating 100 μm (Fig. 4a). The micrograph On the basis of bulk density of surface-adsorbed NE,
of surface-adsorbed NE was not different from colloidal bulk volume 0.61 cm3 was calculated for surface-adsorbed
silicon dioxide indicating uniform adsorption of NE (F3) NE. This bulk volume can be suitably filled in size
over colloidal silicon dioxide (Fig. 4b). No unusual signs “0” capsule having body volume of 0.69 cm3 (32) for a
of precipitation/crystallization of drug/excipient on the sur- dose size of 5 mg of OLM. The in vitro drug release
face of adsorbent were recognizable. This confirms that profile (Fig. 6) of encapsulated surface-adsorbed NE
the NE was homogeneously adsorbed over colloidal sili- showed lower drug release in the initial first hour in
con dioxide. comparison to surface-adsorbed NE attributable to the
additional disintegration step of the capsule shell. Later
on, beyond 3 h, the release magnitude was similar to
Reconstitution of Surface-Adsorbed NE and Evaluation reconstituted NE (RF3) and NE(F3) till the 12th hour. The
similarity factor (f2) of the release profiles was calculated
The particles of surface-adsorbed NE are expected to using PCP Disso V2.08 software. The value of f2 between
be released from the capsule and on contact with body F3(reference) and surface-adsorbed NE (test) was found to
fluids, these should instantaneously re-form into NE quite be 66 and for F3 (reference) and reconstituted NE it was
similar to the initial formulation. To assess the found to be 73. A value of f2 factor between 50 and100
reconstitution abil- ity, specified quantity of surface- indicates similarity in given set of the reference and test
adsorbed NE was shaken with specified quantity of water to samples. Hence, the in vitro drug releases of F3, surface-
get RF3 that was char- acterized for globule size, adsorbed NE and RF3
polydispersity index, zeta poten- tial, and drug content.
Results of the characterization are given in Table IV.
Globule size, polydispersity index, zeta potential, and
percentage drug content of formulation RF3 were found to
be significantly different (p<0.05) in com- parison to
formulation F3. The morphology of the recon- stituted NE
is shown in Fig. 5b. As observed, the globules were
spherical but of slightly bigger in size than F3. The
colloidal silicon dioxide particles might have interfered
with the reconstitution of NE and consequently a higher
globule size, PDI were observed. Lowering of drug content
can be attributed to adsorption of OLM on colloidal silcon
diox- ide. The reconsitutional behavior of surface-adsorbed
NE is expected to be different in vivo. The gastrointestinal
fluid constituents containing biosurfactants in conjugation
with the peristaltic movements will probably facilitate the
recon- stitution of NE favoring lower globule size, probably
Fig. 6. In vitro drug release profiles of F3, surface-adsorbed NE, and
close to initial formulation F3. RF3 in phosphate buffer pH7.4 at 37±1±°C
Surface-Adsorbed NE of Olmesartan Medoxomil
can be adjudged as similar. This study illustrates the
potential of encapsulated surface-adsorbed NE as a
suitable solid unit dosage form to circumvent the handling
limitations associated with SMEDDS or SNEDDS or NE of
water-insoluble therapeutic agents.
CONCLUSION
OLM nanoemulsion was successfully prepared by the
aqueous titration method. Formulation containing capmul
MCM (10% v/v), tween 80 (11.25% v/v), PEG 400 (3.75% v/
v), and double-distilled water (75% v/v) was optimized and
was converted to solid powder by adsorbing onto colloidal
silicon dioxide and encapsulated. The in vitro release profile of
the encapsulated system was statistically similar to
nanoemulsion proving the performance efficacy of the
encapsulated system. However, the performance
characteristics need in vivo evalua- tion. The developed system
can be considered as a novel solid unit dosage form of OLM
nanoemulsion that has the potential to circumvent the handling
limitations associated with NE.
ACKNOWLEDGMENTS
The authors are thankful to Sun Pharmaceutical Indus-
tries, Sikkim, India for providing the gift sample of olmesartan
medoxomil.
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